Non-immunoglobulin based binding agents can be beneficially used in the medical fields of diagnosis, prophylaxis and treatment of diseases. A solution to the disadvantages resulting from antibodies in diagnosis, prophylaxis and treatment of diseases is to provide polypeptides with comparable or even better affinity and specificity towards the specific targets combined with smaller molecular size enabling an improved tissue penetration and thus having better biodistribution properties.
Among non-immunoglobulin-derived small proteins, molecules based on modified ubiquitin are particularly interesting because these molecules promise alternative therapeutic and diagnostic possibilities compared to antibodies. Ubiquitin is a highly conserved, small, single domain protein present in all known eukaryotic cells and is 100% conserved amongst all vertebrates. In addition, ubiquitin naturally occurs in serum lowering the immunogenic potential. This facilitates preclinical development in different species required for toxicological and efficacy studies.
Target-specific ubiquitin muteins are known as Affilin® (registered trademark of Scil Proteins GmbH) proteins (see, for example, EP1626985B1, EP2379581B1, EP2094845B1, and WO2012/172055). Affilin proteins are engineered to generate de novo binding affinity towards desired targets making them ideal for applications that demand targeting and capturing of biomolecules. In addition, Affilin-based molecules can be conjugated with other functional molecules for targeting approaches as well as chemical coupling for half-life modulation (see, e.g. WO2012/172058, WO2014/094799).
It is an objective of the present invention to provide novel binding proteins and methods for the generation and identification of di-ubiquitin muteins with high binding capabilities to a target. It is a further object of the present invention to provide libraries for the identification of novel binding proteins based on di-ubiquitin muteins.
The above-described objectives and advantages are achieved by the subject-matters of the enclosed independent claims. Preferred embodiments of the invention are included in the dependent claims as well as in the following description, examples and figures. The above overview does not necessarily describe all problems solved by the present invention.